Live self-destructing bacterial adjuvants to enhance induction of immunity

ABSTRACT

Disclosed herein are unique adjuvant compositions comprising an attenuated derivative of a bacterial pathogen that undergoes lysis in vivo. In exemplary embodiments, the bacterial pathogen is a Salmonella spp. Also disclosed are methods for enhancing an immune response using the adjuvants disclosed herein.

BACKGROUND

Multicellular organisms have developed two general systems of immunity to infectious agents. The two systems are innate or natural immunity (also known as “innate immunity”) and adaptive (acquired) or specific immunity. The major difference between the two systems is the mechanism by which they recognize infectious agents.

The innate immune system uses a set of germline-encoded receptors for the recognition of conserved molecular patterns present in microorganisms. These molecular patterns occur in certain constituents of microorganisms including: lipopolysaccharides, peptidoglycans, lipoteichoic acids, phosphatidyl cholines, bacteria-specific proteins, including lipoproteins, bacterial DNAs, viral single and double-stranded RNAs, unmethylated CpG-DNAs, mannans and a variety of other bacterial and fungal cell wall components. Such molecular patterns can also occur in other molecules such as plant alkaloids. These targets of innate immune recognition are called Pathogen Associated Molecular Patterns (PAMPs) since they are produced by microorganisms and not by the infected host organism. (Janeway et al. (1989) Cold Spring Hard, Symp, Quant. Biol. 54: 1-13; Medzhitov et al. (1997) Curr. Opin. Immunol. 94: 4-9).

The receptors of the innate immune system that recognize PAMPs are called Pattern Recognition Receptors (PRRs). (Janeway et al. (1989) Cold Spring Harb. Symp. Quant. Biol. 54: 1-13; Medzhitov et al. (1997) Curr. Opin. Immunol. 94: 4-9). These receptors vary in structure and belong to several different protein families. Some of these receptors recognize PAMPs directly (e.g., CD14, DEC205, collectins), while others (e.g., complement receptors) recognize the products generated by PAMP recognition. Members of these receptor families can, generally, be divided into three types: 1) humoral receptors circulating in the plasma; 2) endocytic receptors expressed on immune-cell surfaces, and 3) signaling receptors that can be expressed either on the cell surface or intracellularly. (Medzhitov et al. (1997) Curr. Opin. Immunol. 94: 4-9; Fearon et al. (1996) Science 272: 50-3).

Cellular PRRs are expressed on effector cells of the innate immune system, including cells that function as professional antigen-presenting cells (APC) in adaptive immunity. Such effector cells include, but are not limited to, macrophages, dendritic cells, B lymphocytes and surface epithelia. This expression profile allows PRRs to directly induce innate effector mechanisms, and also to alert the host organism to the presence of infectious agents by inducing the expression of a set of endogenous signals, such as inflammatory cytokines and chemokines, as discussed below. This latter function allows efficient mobilization of effector forces to combat the invaders.

In contrast, the adaptive immune system, which is found only in vertebrates, uses two types of antigen receptors that are generated by somatic mechanisms during the development of each individual organism. The two types of antigen receptors are the T-cell receptor (TCR) and the immunoglobulin receptor (IgR), which are expressed on two specialized ceil types, T-lymphocytes and B-lymphocytes, respectively. The specificities of these antigen receptors are generated at random during the maturation of lymphocytes by the processes of somatic gene rearrangement, random pairing of receptor subunits, and by a template-independent addition of nucleotides to the coding regions during the rearrangement.

Recent studies have demonstrated that the innate immune system plays a crucial role in the control of initiation of the adaptive immune response and in the induction of appropriate cell effector responses. (Fearon et al. (1996) Science 272: 50-3; Medzhitov et al. (1997) Cell 91: 295-8). Indeed, it is now well established that the activation of naive T-lymphocytes requires two distinct signals: one is a specific antigenic peptide recognized by the TCR, and the other is the so called co-stimulatory signal, B7, which is expressed on APCs and recognized by the CD28 molecule expressed on T-cells. (Lenschow et al. (1996) Annu, Rev. Immunol. 14: 233-58). Activation of naive CD4⁺ T-lymphocytes requires that both signals, the specific antigen and the B7 molecule, are expressed on the same APC. If a naive CD4 T-cell recognizes the antigen in the absence of the B7 signal, the T-cell will die by apoptosis. Expression of B7 molecules on APCs, therefore, controls whether or not the naive CD4 T-lymphocytes will be activated. Since CD4 T-cells control the activation of CD8 T-cells for cytotoxic functions, and the activation of B-cells for antibody production, the expression of B7 molecules determines whether or not an adaptive immune response will be activated.

Recent studies have also demonstrated that the innate immune system plays a crucial role in the control of B7 expression. (Fearon et al. (1996) Science 272: 50-3; Medzhitov et al. (1997) Cell 91: 295-8). As mentioned earlier, innate immune recognition is mediated by PRRs that recognize PAMPs. Recognition of PAMPs by PRRs results in the activation of signaling pathways that control the expression of a variety of inducible immune response genes, including the genes that encode signals necessary for the activation of lymphocytes, such as B7, cytokines and chemokines. (Medzhitov et al. (1997) Cell 91: 295-8; Medzhitov et al. (1997) Nature 388: 394-397). Induction of B7 expression by PRR upon recognition of PAMPs thus accounts for self/nonself discrimination and ensures that only T-cells specific for microorganism-derived antigens are normally activated. This mechanism normally prevents activation of autoreactive lymphocytes specific for self-antigens.

Receptors of the innate immune system that control the expression of B7 molecules and cytokines have recently been identified. (Medzhitov et al. (1997) Nature 388; 394-397; Rock et al. (1998) Proc. Natl. Acad. Sci. USA, 95: 588-93). These receptors belong to the family of Toll-like receptors (TLRs), so called because they are homologous to the Drosophila Toll protein which is involved both in dorsoventral patterning in Drosophila embryos and in the immune response in adult flies. (Lemaitre et al. (1996) Cell 86: 973-83). In mammalian organisms, such TLRs have been shown to recognize PAMPs such as the bacterial products LPS, peptidoglycan, and lipoprotein. (Schwandner et al. (1999) J. Biol. Chem. 274: 17406-9; Yoshimura et al. (1999) J. Immunol. 163: 1-5; Aliprantis et al. (1999) Science 285: 736-9).

Vaccines have traditionally been used as a means to protect against disease caused by infectious agents. However, with the advancement of vaccine technology, vaccines have been used in additional applications that include, but are not limited to, control of mammalian fertility, modulation of hormone action, and prevention or treatment of tumors.

The primary purpose of vaccines used to protect against a disease is to induce immunological memory to a particular microorganism. More generally, vaccines are needed to induce an immune response to specific antigens, whether they belong to a microorganism or are expressed by tumor cells or other diseased or abnormal cells. Division and differentiation of B- and T-lymphocytes that have surface receptors specific for the antigen generate both specificity and memory.

In order for a vaccine to induce a protective immune response, it must fulfill the following requirements: 1) it must include the specific antigen(s) or fragment(s) thereof that will be the target of protective immunity following vaccination; 2) it must present such antigens in a form that can be recognized by the immune system, e.g., a form resistant to degradation prior to immune recognition; or it must deliver a DNA vaccine encoding such antigens that will be synthesized by the vaccinated host, stable against degradation and be presentable to be recognized by the immune system and 3) it must activate APCs to present the antigen to CD4⁺ T-cells, which in turn induce B-cell differentiation and other immune effector functions.

Conventional vaccines contain suspensions of attenuated or killed microorganisms, such as viruses or bacteria, incapable of inducing severe infection by themselves, but capable of counteracting the unmodified (or virulent) species when inoculated into a host. Usage of the term has now been extended to include essentially any preparation intended for active immunologic prophylaxis (e.g., preparations of killed microbes of virulent, strains or living microbes of attenuated (variant or mutant) strains; microbial, fungal, plant, protozoan, or metazoan derivatives or products; synthetic vaccines). Examples of vaccines include, but are not limited to, cowpox virus for inoculating against smallpox, tetanus toxoid to prevent tetanus, whole-inactivated bacteria to prevent whooping cough (pertussis), polysaccharide subunits to prevent streptococcal pneumonia, and recombinant proteins to prevent hepatitis B.

Although attenuated vaccines are usually immunogenic, their use has been limited because their efficacy generally requires specific, detailed knowledge of the molecular determinants of virulence. Moreover, the use of attenuated pathogens as vaccines is associated with a variety of risk factors that may compromise their safety for individuals to be vaccinated. Even more troublesome is the general experience since the initial attenuation of pathogens as vaccines by Pasteur is that as one introduces attenuating mutations there is a concomitant decrease in immunogenicity. This is because attenuating mutations decrease the ability of the attenuated vaccine to colonize the vaccinated host or to replicate and persist in that host in lymphoid tissues needed to induce the needed immune responses necessary to confer protective immunity.

The problem with synthetic vaccines (subunit, killed/inactivated pathogens, etc.), on the other hand, is that while they are generally safe they are often non-immunogenic or non-protective. This is because the immunogenic components are limited by the amount introduced in the vaccine at the time of vaccination and these components might be degraded and not persist for a long enough period of time to induce effective immune responses.

Because of these limitations in immunogenicity of many synthetic and attenuated vaccines, it is advantageous to augment that immunity by the co-administration of adjuvants with the vaccine at the time of vaccination. Adjuvants act in a variety of ways, such as by prolonging the stability and presence of the vaccine in host tissues or my stimulating the host immune system to produce cytokines and chemokines to recruit cells of the immune system to the site of vaccination or to stimulate components of the innate immune system to augment vaccine recognition and enhancement of induced immune responses. Unfortunately, there are very few adjuvants used with human vaccines due to safety concerns and the need to validate safety and efficacy with a particular vaccine in clinical trials. Consequently, vaccines are often administered without adjuvants or use alum that is safe but of limited effectiveness. More recently, mono-phosphoryl lipid A (MPLA) has been developed as a safe adjuvant to recruit innate immunity via interaction with TLR4 in a non-inflammatory manner.

An adjuvant is defined as any substance that increases the immunogenicity of admixed antigens. Although certain chemicals are often considered to be adjuvants, they are in effect akin to carriers and are likely to act by stabilizing antigens and/or promoting their interaction with antigen-presenting cells. The best adjuvants are those that mimic the ability of microorganisms to activate the innate immune system. Pure antigens do not induce an immune response because they fail to induce the costimulatory signal (e.g., B7.1 or B7.2) necessary for activation of lymphocytes. Thus, a key mechanism of adjuvant activity has been attributed to the induction of costimulatory signals by microbial, or microbial-like, constituents carrying PAMPs that are routine constituents of adjuvants. (Janeway et al. (1989) Cold Spring Harb. Symp. Quant. Biol., 54: 1-13). As discussed above, the recognition of these PAMPs by PRRs induces the signals necessary for lymphocyte activation (such as B7) and differentiation (effector cytokines). Adjuvants currently typically used for vaccines in humans include Alum and mono-phosphoryl lipid A (MPLA).

Adjuvants are often used in molar excess of antigens and thus can trigger an innate immune response in many cells that do not come in contact with the target antigen. This non-specific induction of the innate immune system to produce the signals that are required for activation of an adaptive immune response can lead to an excessive inflammatory response that renders many of the most potent adjuvants clinically unsuitable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plasmid maps. (A) Lysis vector pG8R111, pBR ori; pYA4589 p15A on; and pYA4595 pSC101 ori. (B) Lysis vectors with improved T2SS bla SS pG8R112, pSC101 on; pG8R113, p15A on, and pG8R114, pBR ori. (C) Lysis vector with T3SS SopE N-80 pG8R110, p15A ori.

FIG. 2. Induction of TLR4, NOD1 and NOD2 signaling by Salmonella strains (χ9052 and χ12499) on HEK-Blue-mTLR4/mNOD1/mNOD2 cell lines. HEK-Blue cell lines, expressing mTLR4, mNOD1 or mNOD2, were stimulated with χ9052 and χ12499. TLR/NOD activation was measured by SEAP activity after incubation of HEK-Blue-mTLR4 (A), HEK-Blue-mNOD1 (B) or HEK-Blue-mNOD2 (C) cells with χ9052 and χ12499. MDP (100 ng/ml) or LPS (100 ng/ml) were used as positive controls. χ9052 and χ12499 were grown in LB broth with D-alanine+DAP+arabinose, sedimented at room temperature, washed with BSG and suspended in tissue culture medium. MDP—muramyl dipeptide

FIG. 3. Survival of χ12517 and χ12518 after inoculation into non-permissive growth conditions. X12517 and χ12518 were grown in unpurple broth with DAP+D-alanine or unpurple broth with arabinose up to OD 0.9 bacterial cells were harvested and resuspended with PBS to desire concentration. Diluted bacteria were inoculated in un-purple broth for the lysis study. In every hour culture were plated on LB agar plates with DAP+D-alanine or LB agar plates with only arabinose and bacteria were counted

FIG. 4. Swimming motility phenotypes of different S. typhimurium mutant strains. Bacterial suspension was spotted onto the middle of the supplemented LB plates with 0.3% agar and incubated at 37° C. for 7 h. The diameter of the colonies was measured in centimeters.

FIG. 5. Activation of TLR4 displayed on HEK cells by different S. typhimurium mutant strains

FIG. 6. Activation of TLR5 displayed on HEK cells by different S. typhimurium mutant strains. HEK-Blue™ mTLR5 cells were stimulated with various Salmonella strains at different MOI of 10 or 1 or 0.01 or 0.001. After 24 h incubation, NF-kB-induced SEAP were determined by reading the OD at 650 nm. The response ratio was calculated by dividing the OD at 650 nm for the treated cells by the OD at 650 nm for the untreated cells.

FIG. 7. Activation of Nod1 and Nod2 present in HEK cells by different S. typhimurium mutant strains

FIG. 8. Enhanced production of antibody production against Ova by co-administration of Family 1 Salmonella adjuvant strain χ9052. 5×106 CFU of χ9052 s.c.; 100 μg Ovalbumen s.c.; 50 μl Alum s.c.

FIG. 9. Mycobacterium tuberculosis H37Rv titers in spleens of mice vaccinated with Mycobacterium bovis Bacille Calmette-Guerin (BCG) with and without inoculation with ENIIRA strain χ12499 or with χ12499 alone.

FIG. 10. Mycobacterium tuberculosis H37Rv titers in spleens of mice vaccinated with M. bovis BCG alone or with PIESV χ12068(pYA4891) or with ENIIRA strains χ12517 or χ12518.

FIG. 11. Mycobacterium tuberculosis H37Rv titers in lungs of mice vaccinated with M. bovis BCG and/or χ12068(pYA4891) with or without inoculation with ENIIRA strain χ12518.

DETAILED DISCLOSURE

Successful pathogens have evolved to either infect the host in a stealth mode to be undetectable and/or suppress, modulate or circumvent induction of immunity, synthesize subterfuge antigens that induce immune responses that confer no protective immunity and/or devise means to colonize and persist in the host. Salmonella vaccine vectors have been continuously modified to eliminate these means as they are discovered and characterized. In addition, other modifications to enhance an induction of innate immune response in the absence of excess inflammation have been made with Salmonella vaccine vectors. Additionally, because live bacterial vaccines have the potential to persist in the environment if shed, a method to solve this problem has been devised. This method achieves regulated delayed lysis and thereby ensures that viable vaccine cells do not persist in vivo or survive if shed into the environment.

Based on these attributes and the observation that Salmonella vaccine vector strains with these properties are entirely safe when administered to two-hour old mice or to pregnant mice or to protein-malnourished mice or to immunodeficient SCID mice, Salmonella strains with some of these properties are used herein to design strains with unique attributes to enhance their safety and efficacy when used as adjuvants.

Vaccines for the prevention of multiple infections caused by many pathogens are nonexistent. Currently existing vaccines often only provide partial protective immunity, or, require repeat vaccinations to provide adequate immunity to a subject. Recombinant attenuated Salmonella vaccine (RASV) vectors for synthesizing and delivering protective antigens encoded by genes from pathogens have been studied. In contrast to the RASV vectors previously described, empty vector control strains which do not deliver a protective antigen invariably but surprisingly provide low levels of protective immunity to a challenge pathogen. While this low level may exceed the level of immunity conferred by buffered saline, this level was significantly less than in animals receiving vaccine strains delivering protective antigens. Consequently, this low-level protective immunity against bacterial, viral and parasitic pathogens occurs as a result of vaccinating animal hosts with empty vector control RASVs.

Consequently, the discovery of and improvements provided herein of live self-destructing Salmonella strains for serving as potent adjuvants (Sal-Adj) for enhancing induction of protective immunity by subunit and live ineffectual vaccines is paramount for the production of universal adjuvant strains. This discovery and the ensuing improvements enables use of Sal-Adj constructs to induce low-level protective immunity to challenge of unvaccinated animals and humans to various bacterial, viral and parasitic pathogens. Because of the principal means by which these Sal-Adj strains exert their beneficial activities (as determined in the studies conducted), they are also referred to as ENhanced Innate Immunity Response Activators (ENIIRAs). Optimal doses, routes and times of administration to enhance induction of protective immunity against infection and persistence by pathogens will be elucidated herein. In a similar way, the RASV vector strains now improved with features to diminish their abilities to subvert induction of immunity and increasing their immunogenicity by enhancing stimulation of innate immunity are now termed Protective Immunity Enhanced Salmonella Vaccine (PIESV) vectors.

Definitions

As used herein the specification, “a” or “an” may mean one or more, unless clearly indicated otherwise. As used herein in the claims, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps.

The term “consisting essentially of” when used in conjunction with adjuvant containing compositions described herein refers to a composition comprising a Sal-Adj or ENIIRA strain and a pharmaceutical carrier without any other immune response enhancing components.

The term “agent” as used herein refers to either adjuvant and/or vaccine.

As used herein, the term “adjuvant” refers to an agent that stimulates and/or enhances an immune response in a subject. An adjuvant can stimulate and/or enhance an immune response in the absence of an antigen and/or can stimulate and/or enhance an immune response in the presence of an antigen. Exemplary embodiments of adjuvants disclosed herein are ENIIRA.

The term “administering” or “administration” of an agent as used herein means providing the agent to a subject using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Agents described herein may be administered by oral, intradermal, intravenous, intramuscular, intraocular, intranasal, intrapulmonary, epidermal, subcutaneous, mucosal, or transcutaneous administration.

The term “co-administration” or “co-administering” as used herein refers to the administration of an active agent before, concurrently, or after the administration of another active agent such that the biological effects of either agents overlap.

An “immune response enhancing amount” is that amount of an adjuvant administered sufficient to enhance an immune response of vaccine administration in a subject compared to vaccine administration without adjuvant administration. An immune response enhancing amount can be administered in one or more administrations.

As used herein, the term “immunogen” refers to an antigen that is recognized as unwanted, undesired, and/or foreign in a subject.

A used herein, the term “immune response” includes a response by a subject's immune system to a vaccine. Immune responses include both cell-mediated immune responses (responses mediated by antigen-specific T cells and non-specific cells of the immune system) and humoral immune responses (responses mediated by antibodies present in the plasma lymph, and tissue fluids). The term “immune response” encompasses both the initial responses to an immunogen as well as memory responses that are a result of “acquired immunity.”

As used herein, the phrase “stimulating or enhancing an immune response” refers to an increase in an immune response in the subject following administration of a vaccine with an adjuvant of the disclosed embodiments relative to the level of immune response in the subject when a vaccine has been administered without an adjuvant.

As used herein, the term “vaccine” refers to an immunogen or a composition comprising an immunogen that elicits an endogenous immune response in a subject (e.g., a human or animal). The endogenous immune response may result in, for example, the switching of a Th1 biased immune response to a Th2 biased immune response, the activation or enhancement of T effector cell responses and/or the reduction of T regulatory cell response, the activation of antigen-specific naive lymphocytes that may then give rise to antibody-secreting B cells or antigen-specific effector and memory T cells or both, and/or the direct activation of antibody-secreting B cells.

The term “pharmaceutically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the successful delivery of the pharmaceutical composition of the Sal-adj strains disclosed herein. As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990). Examples of liquid carriers include, but are not limited to, water, saline, dextrose, glycerol, ethanol and mixtures thereof. The term “protective immunity” as used herein refers to induction of an immune response upon administration of a vaccine sufficient to confer protection against a pathogen.

Overview and Preliminary Studies 1. Bacterial Strains for Adjuvant Compositions.

Many S. typhimurium strains with individual and combinations of deletion and deletion-insertion mutations have been isolated/constructed and all the suicide vectors for these constructions are available to move these mutations into strains to create new Sal-Adj/ENIIRA strains.

Table 1 lists the mutations and their associated phenotypic attributes that were used in these studies. Based on our prior results and the considerations discussed above, we began with three parental strains that exhibit lysis in vivo but with different periods of time needed for lysis and will therefore disperse into tissues away from the inoculation site to different extents. All these strains exhibit complete biological containment features being unable to persist in vivo or survive if released into the environment.

Family A: χ9052 Δalr-3 ΔdadB4 ΔasdA33—requires D-alanine (Salmonella has two alanine racemases) and diaminopimelic acid (DAP) that are unique essential constituents of peptidoglycan that provides the rigid layer of the bacterial cell wall. D-alanine and DAP are only synthesized by bacteria and are totally absent in animal tissues.

Family B: χ12499 Δalr-3 ΔP_(dadB66)::TT araC P_(araBAD) dadB ΔP_(asdA55)::TT araC P_(araBAD) asd—requires presence of arabinose since synthesis of D-alanine and DAP are totally dependent on arabinose-induced synthesis of the dadB-encoded alanine racemase and the asdA-encoded aspartate semialdehyde dehydrogenase. Arabinose is absent in animal tissues but this strain undergoes several cell divisions in vivo until the dadB- and asdA-encoded enzymes are diluted by cell division so that D-alanine and DAP synthesis are insufficient to maintain peptidoglycan integrity.

Family C: χ11730 ΔP_(murA25)::TT araC P_(araBAD) murA ΔasdA27::TT araC P_(araBAD) c2 Δ(wza-wcaM)-8 ΔrelA198::araC P_(araBAD) lacI TT—must be used with a lysis plasmid that also has araC P_(araBAD) regulation of the murA and asdA genes (see next section) to yield a strain that is totally dependent on arabinose-induced synthesis of the enzymes needed to synthesize DAP and muramic acid (another unique essential constituent of peptidoglycan). Depending on the complementing plasmid copy number used, this strain will disseminate more widely and attain higher titers in animal tissues prior to onset of lysis than strains in Families A and B.

The various mutations that are being used to conduct studies to determine the optimal means to stimulate innate immunity that is not excessively inflammatory are listed in Table 1 along with the mutations present in the Family A, B and C starting strains.

TABLE 1 Mutations and associated phenotypes in S. Typhimurium adjuvant strains^(a) Genotype Phenotype A. Deletion and deletion-insertion mutations to facilitate regulated delayed lysis in vivo ΔP_(murA)::TT araC P_(araBAD) murA makes synthesis of MurA, the first enzyme in the synthesis of muramic acid, dependent on arabinose in growth medium and ceases synthesis in vivo due to absence of arabinose (1, 2). MurA decreases due to cell division in vivo to ultimately cause lysis and death (2). The murA defect is complemented by MurA⁺ plasmid vectors (1). ΔmurA mutations are lethal since the product of the gene is phosphorylated that precludes its uptake by Salmonella cells. ΔasdA encodes aspartate semialdehyde dehydrogenase essential for synthesis of diaminopimelic acid (DAP) necessary for peptidoglycan synthesis (3). ΔP_(asdA)::TT araC P_(araBAD) asdA makes synthesis of AsdA dependent on presence of arabinose Δalr encodes one of two alanine racemases essential for synthesis of D- alanine necessary for peptidoglycan synthesis (4). ΔdadB encodes one of two alanine racemases essential for synthesis of D- alanine necessary for peptidoglycan synthesis (4). ΔP_(dadB66)::TT araC P_(araBAD) dadB makes synthesis of DadB dependent on presence of arabinose Δ(wza-wcaM) eliminates twenty enzymes needed to synthesize several exopolysaccharides that promote biofilm formation facilitating persistence and synthesis of GDP-fucose required for colanic acid synthesis (5), which protects cells undergoing cell wall-less death from lysing (6). These exopolymers are also immunosuppressive. ΔrelA the relA mutation uncouples growth regulation from a dependence on protein synthesis, an important attribute in strains with regulated delayed lysis (7, 8) B. Mutations enabling regulation of genes that might be present on plasmid vectors in conjunction with strains undergoing regulated delayed lysis in vivo P_(trc) a promoter expressed at high level under both anaerobic and aerobic conditions and repressed by LacI (9, 10) ΔrelA::araC P_(araBAD) lacI TT the arabinose-dependent synthesis of the LacI repressor is to enable a regulated delayed expression of DNA sequences under the control of P_(trc) (11) ΔasdA::TT araC P_(araBAD) c2 the Asd enzyme is essential for the synthesis of DAP required for peptidoglycan synthesis (12). The arabinose-dependent synthesis of the C2 repressor enables a regulated delayed expression of DNA sequences under control of C2 repressed promoters (1). The ΔasdA mutation is complemented by Asd⁺ plasmids (13). Phage P22 P_(R) promoter is repressible by arabinose-dependent synthesis of the C2 repressor (14) C. Mutations altering synthesis of LPS components ΔpagP::P_(lpp) lpxE mutation causes regulated delayed in vivo synthesis of the codon-optimized lpxE gene from Francisella tularensis to cause synthesis of the non- toxic adjuvant form of LPS lipid A monophosphoryl lipid A (MPLA)(15). The pagP mutation also eliminates a means by which Salmonella alters LPS lipid A in vivo to decrease recruitment of innate immunity by interaction with TLR4 (16) ΔpagL eliminates a means by which Salmonella alters LPS components in vivo to decrease recruitment of innate immunity by interaction with TLR4 (16) ΔlpxR eliminates another means by which Salmonella alters LPS components in vivo to decrease recruitment of innate immunity by interaction with TLR4 (16) ΔarnT eliminates a means by which Salmonella alters LPS lipid A in vivo to decrease recruitment of innate immunity by interaction with TLR4 (Ref) ΔeptA eliminates a means by which Salmonella alters LPS lipid A in vivo to decrease recruitment of innate immunity by interaction with TLR4 (Ref) ΔwaaC eliminates an enzyme needed to synthesize LPS core resulting in a deep-rough phenotype and also renders Salmonella totally attenuated (Q. Kong IAI 2011) ΔwaaG eliminates an enzyme needed to synthesize LPS core resulting in a medium-rough phenotype and also renders Salmonella totally attenuated (Q. Kong IAI 2011) ΔwaaL eliminated the enzyme that joins the LPS O-antigen chain to the LPS core resulting in a moderate-rough phenotype and also renders Salmonella totally attenuated (Q. Kong I&I 2011) D. Mutations altering synthesis of flagellar components ΔfliC180 a specific mutation in the fliC gene encoding the Phase I flagellin protein (17) that contains the TLR5 receptor and a CD4 T-cell epitope. The mutation prevents synthesis of flagella. ΔfliC2426 deletes fliC gene to eliminate synthesis of Phase I flagellin (17) ΔfljB217 deletes fljB gene to eliminate synthesis of Phase II flagellin (17) Δ(hin-fljBA) deletes the sequence necessary for phase switching of flagellin synthesis and eliminates synthesis of the phase II flagellin and the repressor of the fliC gene E. Mutations altering synthesis of fimbrial components ΔP_(stc)::P_(murA) stc causes constitutive synthesis of the in vivo expressed Stc fimbriae that contribute to immunogenicity (18) ΔstcABCD eliminates synthesis of Stc fimbriae (18) ΔP_(saf)::P_(murA) saf causes constitutive synthesis of the in vivo expressed Saf fimbriae that contribute to immunogenicity (18) ΔsafABCD eliminates synthesis of Saf fimbriae (18) F. Mutations eliminating or diminishing effective immunogenicity ΔsifA enables Salmonella to escape the SCV for lysis in cytosol (19) and eliminates a means of immunosuppression (20) ΔsteE G. Mutations leading to degradation of DNA within Salmonella cells ΔrecA enhances rate of DNA digestion in Salmonella cells as a consequence of recombination to liberate DNA fragments with CpG sequences and also renders Salmonella totally attenuated (Ref) ^(a)Δ = deletion; TT = transcription terminator; P = promoter

Table 2 lists the suicide plasmids used to move the mutations including deletion and deletion-insertion mutations listed and described in Table 1 into the Sal-Adj/ENIIRA strains constructed including the Family A, B and C strains listed above and their derivatives described in following sections and Examples as well as in strains yet to be constructed.

TABLE 2 Suicide vectors for constructing the mutations in Table 1 Genotype Suicide Vector Marker A. Deletion and deletion-insertion mutations to facilitate regulated delayed lysis in vivo ΔP_(murA)::TT araC P_(araBAD) murA pYA4686 Cm ΔasdA pYA3736 Cm ΔP_(asdA)::TT araC P_(araBAD) asdA pG8R71 Cm Δalr pYA3667 Cm ΔdadB pYA3668 Cm ΔP_(dadB66)::TT araC P_(araBAD) dadB pG8R73 Cm Δ(wza-wcaM) pYA4368 Cm ΔrelA pYA3679 Cm B. Mutations enabling regulation of genes that might be present on plasmid vectors in conjunction with strains undergoing regulated delayed lysis in vivo ΔrelA::araC P_(araBAD) lacI TT pYA4064 Cm ΔasdA::TT araC P_(araBAD) c2 pYA4138 Cm C. Mutations altering systhesis of LPS components ΔpagP::P_(lpp) lpxE pYA4295 Cm ΔpagL pYA4284 Cm ΔlpxR pYA4287 Cm ΔarnT pYA4286 Cm ΔeptA pYA4283 Cm ΔwaaC pYA5473 Cm ΔwaaG pYA4896 Cm ΔwaaL pYA4900 Cm D. Mutations altering synthesis of flagellar components ΔfliC180 pYA3729 Cm ΔfliC2426 pYA3702 Cm ΔfljB217 pYA3548 Tet Δ(hin-fljBA) pG8R306 Cm E. Mutations altering synthesis of fimbrial components ΔP_(stc)::P_(murA) stc pYA5053 Cm ΔstcABCD pYA5007 Tet ΔP_(saf)::P_(murA) saf pYA5055 Cm ΔsafABCD pYA4586 Tet F. Mutations eliminating or diminishing effective immunoigenicity ΔsifA pYA3716 Cm ΔsteE G. Mutations leading to degradation of DNA within Salmenella cells ΔrecA pYA4680 Cm ^(a) Δ = deletion; TT = transcription terminator; P = promoter 2. Plasmids for Adjuvant Strains with Regulated Delayed Lysis In Vivo. Family C

Sal-Adj strains will be used in conjunction with plasmids conferring a regulated delayed lysis in vivo phenotype (FIG. 1). This phenotype is due to the araC P_(araBAD)-regulated murA and asd genes with GTG start codons to decrease translation efficiency and the P22 P_(R) (located in opposite orientation to transcription of the araC P_(araBAD) GTG-murA GTG-asd genes) that is repressed by the C2 repressor encoded in the ΔsdA27::TT araC P_(araBAD) c2 mutation. MurA, AsdA and C2 are synthesized when Family C strains are grown with arabinose but cease to be synthesized in vivo. Thus, as C2 concentration decreases due to cell division in vivo, P_(R)-directed anti-sense mRNA synthesis commences to block translation of residual asdA and murA mRNA. Transcription terminators (TT) flank all plasmid domains for controlled lysis, replication and gene expression so that expression in one domain does not affect activities of another domain. The time of onset of in vivo lysis can be controlled, in part, by using plasmids with different copy numbers. These plasmids, especially those with Type 2 and 3 secretion systems (T2SS; T3SS), can be used to synthesize and deliver different effector molecules such as single and double stranded RNA, flagellins, pilins, lipo-proteins or to enable synthesis and delivery of macromolecules such as teichoic acid, lipo-teichoic acid, mannan, etc. to enhance induction of innate immune responses.

3. Observations with Unexpected Ability of Empty Vector Control RASV Strains to Confer Low-Level Protective Immunity to Pathogens or Decreased Ability of Pathogens to Multiply in Hosts or Reduce Performance.

As stated above, we have observed, in developing RASV/PIESV vectored vaccines, that the empty vector control groups (having a vector plasmid not encoding a protective antigen) invariably had higher survival or performance after challenge than the control groups receiving buffered saline (BS). This was especially true with vaccine vector strains that undergo lysis in various cell compartments in vivo. This implied that we might be recruiting innate immunity via activation of internal Nod and TLR9 receptors by release of peptidoglycan components and DNA intracellularly. We present results from some of these studies in Table 3.

TABLE 3 Empty vector PIESVs and protective immunity* Percent Survival Pathogen RASV Path challenge BS PIESV − Ag PIESV + Ag Influenza PIESV-Flu Influenza WSN Av (3) 16 29 90 Yersinia pestis PIESV-Yp Y. pestis (s.c.) Av (3) 0 38 83 S. pneumoniae PIESV-Sp S. pneumoniae Av (7) 0 5 61 M. tuberculosis PIESV-Mtb M. tuberculosis Empty vector control reduced Mtb colonization more than BS (3 comparisons) C. perfringens RASV-Cp C. perfringens Empty vector reduced lesions & mortality, enhanced feed conversion & weight gain more than BS (4 comparisons) E. tenella PIESV-Eimeria Eimeria Empty vector enhanced feed conversion & weight gain more than BS (2 comparisons) *First 4 studies in inbred mice and last 2 studies in outbred chickens The empty vector PIESV strains used for the results presented in Table 3 are very analogous to the Family C ENIIRA strains although some of these strains did not have the regulated delayed lysis in vivo phenotype. 4. Interaction of Family A and B Strains with HEK Cells Displaying TLR and Nod Factors.

To evaluate the ability of Salmonella strains with differing genotypes to stimulate innate immune responses, we have used HEK293 cells with a NFkB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene (InvivoGen) and displaying Nod1, Nod2 and TLR4. We initially determined that all three Family A, B and C strains containing a plasmid encoding GFP were highly invasive into HEK cells. The χ9052 and χ12499 strains were grown in LB broth to maximize expression of the SPI-1 invasion phenotype, sedimented at room temperature and suspended in tissue culture medium. HEK cells at 2×10⁵ cells/ml were mixed with bacterial cells at a MOI of 10 in a volume of 200 μl in 96 well plates. Unattached bacteria were removed by washing in tissue culture medium and then plates were incubated at 37° C. in 5% CO₂ over 24 h with periodic scanning at 650 nm for production of the blue reagent due to secretion of alkaline phosphatase from the HEK cells. FIG. 2 presents the results. χ9052 was best at activating Nod1, whereas both strains activated Nod2 equal to or better than muramyl dipeptide (MDP). Both strains activated TLR4 better than LPS at all times measured.

5. Enhanced NFκB in HEK Cells Displaying TLR5.

S. typhimurium χ9026 with ΔfljB217 ΔfliC180 mutations that overproduces a truncated FliC flagellin having the receptor for TLR5 stimulates significantly higher levels of IL6 and TNFα production in GALT and MLN cells than the non-flagellated χ9028 strain with ΔfljB217 ΔfliC2426 and also stimulates higher levels of NF-kB production in HEK cells displaying the murine TLR5. Expansion of the data in analyzing interaction of Sal-Adj/ENIIRA strains with HEK cells is presented in the Examples.

EXAMPLES Example 1. Materials and Methods Bacterial Strains, Media and Bacterial Growth.

All Sal-Adj/ENIIRA strains as well as other strains possessing individual mutations used in strain constructions are derived from the highly virulent S. typhimurium UK-1 strain χ3761 (21). LB broth and agar (22) will be used as complex media for propagation and plating of Salmonella strains. Purple broth (PB) (Difco), which is devoid of arabinose (Ara), was also used since LB contains low levels of arabinose. MacConkey agar with 0.5% lactose (Lac) and 0.1% Ara were used to enumerate bacteria recovered from mice. Mycobacterium tuberculosis H37Rv was propagated in Middlebrook 7H9 broth (Difco) supplemented with 10% albumin, dextrose and sodium chloride (ADS). Middlebrook 7H11 agar, supplemented with 10% ADS was used to determine bacterial CFU titers in the lungs and spleens of immunized mice challenged with M. tuberculosis. Bacterial growth was monitored spectrophotometrically and by plating for colony counts. S. typhimurium PIESV strain χ12068 carrying the plasmid pYA4891 was used in two of the experiments described in Example 5. χ12068 has the following genotype: (ΔP_(murA25)::TT araC P_(araBAD) murA ΔasdA27:TT araC P_(araBAD) c2 Δpmi-2426 Δ(wza-wcaM)-8 ΔrelA197::araC P_(araBAD) lacI TT ΔrecF126 ΔsifA26 ΔwaaL46 ΔpagL12:TT araC P_(araBAD) waaL), which has the regulated delayed lysis in vivo phenotype and escapes the SCV to lyse in the cytosol to deliver antigens to the proteosome for class I presentation to induce CD8+, CD 17+ and NKT-dependent immune responses. The plasmid pYA4891 is designed for use in regulated delayed lysis in vivo PIES Vs and is a derivative of pYA4589, described in FIG. 1. Plasmid pYA4891 carries genes encoding three M. tuberculosis antigens: Ag85A, ESAT-6 and CFP-10 (31). Ag85A is one of three highly conserved mycolyl transferases integral to the synthesis of mycolic acids, an essential component of the mycobacterial cell envelope. Antigens 85A, B and C transport mycolyl residues from the mycobacterial cytoplasm to the growing chains of mycolic acids (38). The antigens ESAT-6 and CFP-10 of M. tuberculosis are two secreted proteins that are important virulence factors and are two of the most protective antigens of M. tuberculosis (39-41). M. tuberculosis ESAT-6 and its homologue TB10.4 are components of two subunit vaccines against M. tuberculosis that have been shown to be protective in immunized mice (31,41) and guinea pigs (41) and are currently in Phase Ha human clinical trials (42). In χ12068(pYA4891), Ag85A is exported from χ12068 via the Salmonella Type 2 secretion system during in vivo growth; ESAT-6 and CFP-10 are delivered from χ12068 by the Salmonella Type 3 secretion system directly into host cell cytoplasm during in vivo growth. All three antigens are delivered into the cytoplasm of host cells upon lysis of χ12068, following regulated delayed lysis.

Molecular and Genetic Procedures

Methods for DNA isolation, restriction enzyme digestion, DNA cloning and use of PCR and real-time PCR for construction and verification of bacterial strains and vectors are standard (23). Defined deletion mutations with and without specific insertions have been constructed for all mutations listed in Table 1 using flanking sequences derived from the S. typhimurium parent χ3761. These mutations are introduced using either phage P22HTint (24, 25) transduction of suicide vectors integrated into the deletion mutation followed by selection for sucrose resistance (26) or by conjugational transfer of suicide vectors using standard methods (27, 28) with the suicide vector donor strains χ7213 and χ7378 (29). Plasmid constructs were evaluated by DNA sequencing and for ability of sugar-regulated sequences to specify synthesis of proteins using gel electrophoresis and western blot analyses (30).

Strain Characterization

Sal-Adj/ENIIRA strains were fully characterized at each step in their construction. Genetic attributes were confirmed by PCR with appropriate primers. Measurement of LPS core and O-antigen were performed after electrophoresis using silver-stained gels (88). This analysis is done after every step in any strain construction to eliminate possible spontaneous variants if they arise. We also validate the complete sensitivity of all Sal-Adj/ENIIRA strains to all antibiotics that might ever be used to treat Salmonella infections. Metabolic attributes are evaluated using API-20E tests. The presence of the recA mutation is determined by inability to undergo recombination using P22 transduction and extreme sensitivity to UV light.

Swimming motility was assessed on LB plates solidified with 0.3% agar and supplemented with appropriate supplements (arabinose or DAP or D-alanine). Strains were grown statically overnight in the appropriate media. The next day, 50 μl of this culture was inoculated into 2 ml of the appropriate media and grown with aeration at 37° C. to an optical density at 600 nm (OD600) of 0.8 to 0.85. One milliliter samples of cultures were pelleted at 4,500×g and were resuspended in 1 ml BSG. Five microliter samples of bacterial suspension were spotted onto the middle of the plates, which were then incubated at 37° C. for 7 h.

Production and secretion of flagellin was determined by shearing appendages from cells in culture using a Waring blender. After removal of cells by centrifugation, proteins in the supernatant were precipitated with TCA and then hydrolyzed by boiling in SDS buffer prior to electrophoresis. Antiserum against FliC and FljB flagellin were used to detect production of the Phase I and Phase II flagellins, respectively, by western blotting.

Production of fimbrial adhesins is determined using antisera to the Saf and Stc fimbrial subunits by western blot analyses after electrophoresis of precipitated protein fractions obtained after shearing bacterial strains in a blender to remove flagellar and fimbrial appendages (as described above).

Measurement of growth and lysis after inoculation of bacteria into media not permissive for growth was evaluated spectrophotometry and by dilution and plate counting to determine viable cell titers. For these evaluations we used Purple broth since it totally lacks the sugars arabinose, mannose and rhamnose that are present at very low concentrations in LB broth. We also use un-Purple broth that just lacked the pH indicator dye. Bacterial strains were grown statically overnight in the appropriate media supplemented with 0.1% arabinose and/or 50 μg DAP and/or 20 μg D-alanine. The next day, 50 μl of this culture was inoculated into 2 ml of the appropriate media and grown with aeration at 37° C. to an optical density at 600 nm (OD600) of 0.8 to 0.85. Cells were then centrifuged twice and washed and resuspended in un-Purple broth at a cell density of about 5×10⁷ CFU/ml and then grown with rotary aeration at 37° C. Absorbancies at OD_(600 nm) were monitored continuously and dilutions and plate counts on permissive agar medium made at 30 and/or 60 min intervals.

Cell Culture Methods and Use of HEK293 Cells to Monitor Initiation of Innate Immune Responses

HEK293 cells with the murine TLR1, TLR2, TLR4, TLR4 MD2 CD14, TLR5, TLR5 CD14, TLR6, TLR9, Nod1 and Nod2 were used with the NF-kB SEAP reporter system to enable read outs at A650 nm. Sal-Adj/ENIIRA strains were grown to maximize their invasiveness and determine bacterial cell attachment to, invasion into and survival in HEK cells and monitor stimulation of NF-kB production by HEK cells over a 24 h period. More specifically, Salmonella strains were grown in LB with appropriate supplements at 37° C. to an optical density at 600 nm (OD600) of 0.8 to 0.9. Bacteria were harvested by centrifugation at 4,500×g at room temperature and were resuspended in endotoxin free water at the densities required to produce the desired dose ENIIRA strain as multiplicity of infection (MOI 10, 1, 01 and 001) relative to HEK cell density in the appropriate volume. Bacterial samples of 20 μl were added per well in a flat-bottom 96-well cell culture plate.

HEK-Blue™ mTLR4 or HEK-Blue™ mTLR5 or HEK-Blue™ mNOD1 or HEK-Blue™ mNOD2 cells were purchased from InvivoGen, San Diego, Calif., USA. The cells were cultured at 37° C. in 5% CO₂ in 25 vented flasks using Eagle's Minimum Essential Medium (EMEM) ATCC® 30-2003™ containing heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin, and normocin (InvivoGen). Cells were grown to 50-70% confluence and were resuspended in HEK-Blue detection media at a cell density 2-5×10⁶ viable cells per milliliter. Cell suspensions of 180 μl were added to the previous bacterial samples added to the 96-well cell culture plate. Plates were incubated at 37° C. in 5% CO₂ for 24 h. SEAP activity was determined using a spectrophotometer at 650 nm. The response ratios were calculated by dividing the OD at 650 nm for the treated cells by the OD at 650 nm for the untreated cells.

Animal Experimentation to Monitor Safety and Efficacy of Sal-Adj/ENIIRA Constructs Administered by Different Routes to Young Adult Mice

Ten-fold dilutions (10⁴-10⁸ CFU) of S. typhimurium χ12517 or χ12518 strains were administered by intravenous (I.V.) or subcutaneous (S.C.) or intranasal (I.N.) routes to 6-week-old female BALB/c mice by following our standard procedures (Q. Kong et al., 2011. Infection and Immunity). Briefly, adjuvant strains were grown statically overnight in the appropriate media. The next day, 100 μl of each culture was inoculated into 5 ml of the appropriate media and grown with aeration at 37° C. to an optical density at 600 nm (OD600) of 0.8 to 0.85. Cultures were pelleted at 4,500×g at room temperature and were resuspended in BSG at the densities required to produce the desired dose in the appropriate volume. A volume of 20 μl of bacterial suspension containing the appropriate dose was inoculated by the various routes at day 0. Mice were monitored for death twice a day up to 3 weeks.

Animal Experimentation to Monitor Safety and Efficacy of Sal-Adj/ENIIRA Constructs to Augment Immune Responses to Ovalbumin (Ova)

Imject Alum was purchased from Thermo Scientific (cat #77161) and ova albumin was purchased from Sigma (A2512-1G). Ova albumin was dissolved in BSG with a stock concentration 2 mg/ml. Adjuvant strain χ9052 was grown statically overnight in LB supplemented with DAP and D-alanine. The next day, 100 μl of this culture was inoculated into 5 ml of the appropriate media and grown with aeration at 37° C. to an optical density at 600 nm (OD600) of 0.8 to 0.9. Bacteria were harvested by centrifugation at 4,500×g at room temperature and were resuspended in BSG at the densities required to produce the desired dose in the appropriate volume. Above suspension of 500 μl was emulsified with 500 μl Imject Alum by repeatedly passing through a syringe needle. Each mouse was inoculated subcutaneously with 100 μl of suspension. Six-week-old BALB/c mice were divided into three groups of five mice each named Group-I [Ova (100 μg)+χ9052 (5×10⁶)], Group-II [Ova (100 μg)] and Group-III [Ova (100 μg)+Imject Alum (50 μl)]. Animals were immunized subcutaneously. Blood was sampled from immunized mice for antibody determination by ELISA at 2, 4 and 6 weeks post-inoculations.

Animal Studies to Determine Ability of Sal-Adj/ENIIRA Constructs to Augment Level of Protective Immunity Against M. tuberculosis (Mtb) Challenge of BCG Immunized Mice

C57/BL6 mice of both sexes were given a s.c. dose of 5×10⁶ CFU of M. bovis BCG Pasteur (ATCC 35734) on day 0 without and with varying doses in CFU of different Sal-Adj constructs by s.c. (1×10⁵ CFU), i.n. (1×10⁷ CFU), and i.v. (5×10⁴ CFU) routes. On day 28, sera were collected from all mice. In addition, two mice from each group were euthanized, livers and spleens were removed, processed and used for flow cytometric analyses. On day 35 all mice were infected with aerosolized M. tuberculosis H37Rv (50 to 100 bacteria per mouse) using a Glas-Col Inhalation Instrumental). Six weeks after challenge, mice were euthanized, lungs and spleens removed aseptically, tissues homogenized, homogenates diluted and samples plated on Middlebrook 7H11+10% ADS agar plates (Difco). Plates were incubated at 37° C. for 3-4 weeks to determine the Mtb CFU in these organs.

Monitoring Immune Responses

Antigen Preparation

Purified Ova was obtained commercially. M. tuberculosis antigens Ag85A, ESAT-6 and CFP-10 were purified as His-tagged proteins from recombinant E. coli. These antigens were used for immunoassays as described below.

ELISA

Serum antibodies were measured in blood collected by submandibular bleeding. We determined the concentrations of IgG and IgA against Ova in μg/ml and the concentrations of IgG against Ag85A, ESAT-6 and CFP-10 in μg/ml. To distinguish between Th1 and Th2 responses, we determined titers of IgG1 and IgG2a for Ova and titers of IgG1 and IgG2b for Ag85A, ESAT-6 and CFP-10. The 96-well plates were coated with 100 ng Ova or with 0.5 to 1 μg of each M. tuberculosis antigen. Free binding sites were blocked with the SEABLOCK Blocking Buffer (Thermo Fisher). Anti-Ova titers or anti-Ag85A, anti-ESAT-6 or anti-CFP-10 titers in the serum (dilution 1:100) were detected with biotinylated goat anti-mouse IgG, IgG1, IgG2a or IgA for Ova and biotinylated goat anti-mouse IgG, IgG1 or IgG2b for Ag85A, ESAT-6 and CFP-10 (Southern Biotechnology) followed by incubation with a streptavidin-alkaline phosphatase conjugate (Southern Biotechnology). Color development (absorbance at 405 nm) with p-nitrophenyl phosphate (Thermo Fisher Scientific) was recorded with an automated ELISA plate reader (EL311SX; Biotek). Unconjugated mouse antibodies (Southern Biotechnology) (IgG, 5 μg/ml to 40 ng/ml; IgG1, IgG2a or IgG2b, 1 μg/ml to 8 ng/ml; IgA 62.5 ng/ml to 0.46 ng/ml) were serially diluted and coated on a 96-well plate in duplicate. Standard curves were generated by plotting the OD₄₀₅ values against the representative concentrations of the diluted unconjugated antibody solutions and fitted to a 4-parameter logistic curve (R2≥0.98). The absorbance values of experimental samples were fit into the standard curve to interpolate antibody concentrations. All samples were analyzed in triplicate. We also used ELISPOT assays (32) in initial studies to determine whether antigen-specific IgA and IgG secreting peripheral blood lymphocytes are induced 10 to 15 days after vaccination with Ova and adjuvants.

Cellular Immune Responses and Flow Cytometry Analyses

These more detailed evaluations of immune responses induced were deferred until optimal Sal-Adj constructs were identified and were conducted in the studies to be proposed in a R01 application pending conduct of the research proposed in this application.

Flow Cytometry

Flow cytometry was used to quantitate populations of antigen-specific CD4⁺ and CD8⁺ T cells and antigen-specific cytokine-secreting cells in the lungs and spleens of mice immunized with M. bovis BCG in combination with Sal-Adj constructs. Lymphocytes were isolated from homogenized lungs and spleens of immunized or PBS control mice by centrifugation of the cell lysates through Percoll gradients. After washing the purified lymphocytes with PBS, the cells were simulated with 10 μg/ml of purified protein antigens for 24 h. Fc blocking reagent was used to prevent non-specific binding of antibodies to Fc receptors on the lymphocytes. Surface staining using anti-mouse fluorophore-labelled antibodies (Biolegend), followed by fixation of the cells with 4% para-formaldehyde was used to identify subsets of lymphocytes. Gating on both CD4 and CD8 was done on the CD3⁺ lymphocyte population to detect the percent of antigen-specific CD4⁺ and CD8⁺ cells expressing effector KLRG1, PD1 or memory CD62L, CD127 molecules. To detect intracellular cytokines, lymphocytes were stained for surface markers CD3, CD44, CD4 and CD8, followed by treatment with BD Sciences Cytofix/Cytoperm and stained intracellularly with combinations of IFN-γ and TNF-α antibodies. All samples were analyzed in the Department of Infectious Diseases and Immunology Flow Cytometry Core Facility on an 8-color FACSCanto, 18-color FACSFortessa flow cytometer with a SH800Z Cell Sorter. Data analyses and statistical comparisons among the samples from immunized and non-immunized mice was done using the Flow JO software.

Splenomegaly.

To determine protection against challenge with M. tuberculosis H37Rv, the spleens and lungs are weighed individually after they are removed from the euthanized mice. The number of CFUs determined from plating samples of homogenized lungs and spleens are reported as CFU per gram of tissue. When spleens were removed from mice immunized with ENIIRA strains, it was immediately evident by visual observation, that some spleens were significantly enlarged, compared to the spleens of unimmunized mice or mice immunized with M. bovis BCG or mice immunized with PIESV χ12068(pYA4891).

Statistical Analyses

All results were analyzed using the most appropriate statistical test from the SAS program to evaluate the relative significance or lack thereof of results obtained.

Example 2. Construction and Characterization of Sal-Adj/ENIIRA Strains a. Introduction

Sal-Adj/ENIIRA strains of the starting genotypes for the Family A, B and C strains were initially compared to determine which most enhances induced immune responses to Ova and protective immunity to M. tuberculosis challenge. This was because it is possible that strains from different families will each be more efficacious in one evaluative test than the other. In these initial studies, the Family A and B strains were found to be most efficacious, in all probability since they invade efficiently and undergo lysis more rapidly. In accord with this, Family C strains that underwent regulated lysis in vivo more rapidly, induced higher innate immune responses than did Family C strains that underwent more cell divisions in vivo prior to lysis. Based on these results we are continuing to develop improved Family C strains as the PIESV vector strains as vaccine constructs for protective antigen and DNA vaccine delivery to prevent infectious diseases. Since recruitment and induction of innate immunity initially upon vaccination would be most beneficial in augmenting induction of acquired immunity by a vaccine, we have focused on developing the Family A and B ENIIRA strains to serve as superior adjuvants.

Comparative evaluation of the survival of Family A and B strains after growth in Purple broth with arabinose and inoculation into medium without arabinose indicated rapid lysis and death of the Family A strain χ9052 whereas the Family B strain χ12499 increased in cell number for several cell divisions before lysis and death commenced (FIG. 3). These results were as expected and suggested that the Family B strains might be more efficacious in inducing innate immune responses due to this short-term proliferation in vivo prior to onset of lysis. While this could be beneficial, it was possible that the Family B strains might also be more inflammatory. These considerations thus guided the design of safe efficacious ENIIRA strains to recruit early onset innate immunity. Sal-Adj/ENIIRA strains derived from the Family A and B strains were made with combinations of genetic modifications to enable determining which mutations were beneficial or were marginal in benefit. This then enabled us to further optimize and enhance induced innate immune responses. The phenotypic properties associated with each mutation in the strains constructed are presented in Table 1 and Table 2 lists the suicide vectors used to introduce the various mutations into the strains constructed.

b. Sal-Adj/ENIIRA Strains Constructed

Table 3 lists the strains constructed from the Family A strain χ9052. The properties associated with the mutations present are described in following sections.

TABLE 3 Family A strain genotypes and derivations Chi number Genotype Parent χ9052 Δalr-3 ΔdadB4 ΔasdA33 χ8901 χ12503 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 χ9052 χ12512 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE χ12503 χ12515 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12512 χ12517 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12515 ΔlpxR9 χ12553 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 Δ(hin fljBA)-219 χ12503 χ12554 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12517 ΔlpxR9 Δ(hin fljBA)-219 χ12555 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12515 ΔwaaC41 χ12556 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12515 ΔwaaG42 χ12557 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12515 ΔwaaL46 χ12558 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12517 ΔlpxR9 ΔwaaC41 χ12559 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12517 ΔlpxR9 ΔwaaG42 χ12560 Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) IpxE ΔpagL7 χ12517 ΔlpxR9 ΔwaaL46

Table 4 lists the strains constructed from the Family 2 strain χ12499. The properties associated with the mutations present are described in following sections.

TABLE 4 Family B strain genotypes and derivations Chi number Genotype Parent χ12499 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12498 χ12504 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12499 ΔfliC180 χ12513 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12504 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE χ12516 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12513 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE ΔpagL7 χ12518 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12516 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE ΔpagL7 ΔlpxR9 χ12542 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12518 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE ΔpagL7 ΔlpxR9 ΔwaaC41 χ12543 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12518 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE ΔpagL7 ΔlpxR9 ΔwaaG42 χ12544 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12518 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE ΔpagL7 ΔlpxR9 ΔwaaL46 χ12545 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12504 ΔfliC180 ΔwaaC41 χ12546 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12504 ΔfliC180 vwaaG42 χ12547 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12504 ΔfliCl80 Δ(hin fljBA)-219 χ12548 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12518 ΔfliC180 ΔpagP81 ::P_(lpp) IpxE ΔpagL7 ΔlpxR9 Δ(hin fljBA)-219 χ12549 ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB χ12504 ΔfliC180 ΔwaaL46

c. Construction of Strains with Alterations in LPS Structure

S. typhimurium is a gram-negative bacterium that contains LPS in its outer membrane. One vital component of LPS is lipid A that has a repeating disaccharide with six attached lipid acyl chains. Lipid A constitutes the potent endotoxin that can cause sepsis and death. The level of sensitivity to this endotoxin varies considerably among animal species with cattle, horses, dogs and humans being far more sensitive than chickens and even mice (34). It is also the lipid A that interacts as an agonist with TLR4 to recruit an innate immune response. However, Salmonella has evolved as a successful pathogen so as to infect more successfully by reducing the ability of its lipid A to trigger innate immunity by modifications that reduce the agonist activity of its lipid A. This is accomplished by decorating the lipid acyl chains with small molecules. Since lysis of ENIIRA strains immediately release the LPS with the lipid A endotoxin, we constructed strains χ12717 (Family A) and χ12518 (Family B) with the ΔpagP81::P_(lpp) lpxE deletion-insertion mutation so that the strain synthesizes the non-toxic adjuvant mono-phosphoryl lipid A rather than the toxic lipid A while retaining its ability to activate TLR4 via the MD2 (rather than MyD88) pathway (33). Inactivation of the pagP gene is important since its gene product modifies lipid A to reduce its agonist activity. The promoter from the E. coli lipoprotein gene (lpp) is one of the strongest promoters since the lipoprotein synthesized is the most abundant protein in gram negative bacteria. It is used here to drive the constitutive expression of the lpxE gene from Francisella tularensis that eliminates a 4′ phosphate decoration of the lipid A disaccharide that is responsible for endotoxicity. This alteration thus precludes excess inflammation due to release of endotoxin in vivo although release of other cell constituents in Family B strains that undergo several cell divisions in vivo prior to onset of lysis might still be too inflammatory at high doses.

We therefore evaluated the relative attenuation/virulence of these two strains in mice by delivery of doses of 10⁴, 10⁵, 10⁶ and 10⁷ CFU by the i.v. route, doses of 10⁵, 10⁶, 10⁷ and 10⁸ CFU by the i.n. and s.c. routes, and 10⁹ CFU by the oral route. All mice survived challenge at all doses by all routes when infected with the Family A strain χ12517. However, some mice died when infected with the Family B strain χ12518 at doses above 10⁶ CFU by the i.v. and i.n. routes and above 10⁷ CFU by the s.c. route, while all mice survived oral inoculation. These results indicated that the several cell divisions in vivo of the Family B strains, due to the regulated delayed lysis in vivo attribute would necessitate animal studies using lower doses and ultimately for the need to introduce additional mutations to preclude excess inflammatory responses leading to mortality.

The ΔpagL7 and ΔlpxR9 mutations were also included to eliminate two additional means that Salmonella uses to lessen recruitment of innate immunity by reducing the agonist activity of lipid A (15). The benefits of these two mutations to enhancing TLR4 recruitment is demonstrated below in studies with HEK cells displaying the TLR 4 ligand. The eptA and arnT mutations (Table 1) have also been included in some ENIIRA strains as they also encode enzymes that lessen the agonist activity of lipid A.

Since some of the Family B derived strains administered at high doses by parenteral routes (i.v., s.c.) were too inflammatory, we constructed strains with deletion mutations in the waaC, waaG and waaL genes that eliminate synthesis of the middle LPS core, outer LPS core and LPS O-antigen, respectively, to confer complete attenuation. Since the waaC and waaG mutations reduce synthesis and display of flagella and might possibly reduce secretion of flagellin that is important for recruiting innate immunity via interaction with TLR5, the use of strains with the waaL mutation is preferred.

d. Construction of Strains with Alterations in Flagella and Flagellin Release

A non-flagellated strain χ9028 with the ΔfliC2426 and ΔfljB217 mutations and strain χ9026 in which the ΔfliC2426 mutation is replaced with the ΔfliC180 mutation to result in synthesis and release of a truncated flagellin that activates TLR5 (see Overview/Preliminary studies) were used in initial evaluations. We initially constructed Family A and B strains with the ΔfliC180 mutation since initial results showed that this mutation enhanced production and release of the FliC flagellin that activates TLR5. In addition, the phase-lock mutation Δ(hin-fljBA)-219 was introduced so that the FliC protein is made by all Sal-Adj/ENIIRA cells. The fljB mutation eliminates the ability of the ENIIRA strains to synthesize the Phase II flagellin and the fljA mutation eliminates synthesis of the repressor that would block transcription of the fliC gene that specifies the Phase I flagellin. A further enhancement in flagellin production was achieved, in one embodiment, by introducing another mutation (cfs) that causes constitutive high-level flagellin synthesis.

As shown in FIG. 4, strains with the ΔfliC180 mutation still display motility if they have a wild-type fljB gene but are unable to exhibit motility when a fljB mutation is present. Nevertheless, all strains with the ΔfliC180 mutation produce secreted flagellin as revealed by western blotting but this flagellin is unable to assemble into functional flagella to convey motility to cells.

e. Construction of Strains with Alterations in Display of Fimbriae

S. typhimurium has 12 operons that specify synthesis of fimbriae (18) and most of these are subject to regulation such that they are only synthesized in certain environments. We recently identified four fimbrial operons using the IVET system to identify in vivo expressed genes that are synthesized in the spleen after oral infection whereas the other eight fimbrial operons were not expressed (18). It was also determined that constitutive synthesis of the in vivo-synthesized Saf and Stc fimbriae significantly enhanced induction of protective immunity of a RASV/PIESV strain (18). Strains will therefore evaluated for interaction with HEK cells and enhancing Ova immune responses lacking the other fimbrial operons expressed in vivo with either the deletion-insertion mutation ΔP_(stc53)::P_(murA) stc or ΔP_(saf55)::P_(murA) saf to cause constitutive synthesis of Stc or Saf fimbriae.

f. Other Constructions

In the initial comparison of the Family A, B and C parental lysis strains, derivatives were compared with the ΔsifA26 mutation that eliminates a means of immunosuppression and enables S. typhimurium cells to escape from the Salmonella-containing vesicle (SCV) such that lysis can occur in the cytosol. In RASV strains that undergo regulated delayed lysis in vivo, escape from the SCV enables lysis of some vaccine cells to occur in the cytosol for delivery of protective antigens to the proteosome for Class I presentation and enhanced induction of CD8-dependent cellular immunity. Although individual cells of ENIIRA strains with the regulated delayed in vivo lysis attribute, can lyse prior to invasion into host cells or after invasion into cells whether contained in the SCV or after escape from the SCV (due to a ΔsifA mutation) or after escape from a host cell undergoing pyroptosis, such lysis will release peptidoglycan components and DNA. Since peptidoglycan components activate Nod1 and Nod2 and CpG containing DNA sequences activate TLR9, the location of the release of these components will impact the level of activation of Nod1, Nod2 and TLR9 that are located internally in host cells. On average, ENIIRA strains with the ΔsifA26 mutation would therefore be expected to better activate Nod1, Nod2 and TLR9.

The recA mutation blocks genetic recombination but about 10 percent of cells at each cell division undergo Rec-less cell death since occasional recombination between replicating chromosomes leads to rapid unregulated degradation of DNA. Consequentially, ENIIRA strains with recA mutations should be more efficient in liberating DNA fragments containing CpG sequences to activate TLR9. An additional benefit is that recA mutants of S. typhimurium are totally avirulent and do not induce disease symptoms when administered to animal hosts. We have therefore inserted the ΔrecA62 mutation into candidate ENIIRA strains.

Some Salmonella pathogenicity island (SPI) 2 genes are effectors that can dampen induction of innate immune responses. The steE gene seems to be in this category such that ENIIRA strains unable to synthesize the steE gene product are investigated for impact on induction of innate immune responses using the HEK cell lines.

g. Characterization of Constructed ENIIRA Strains

All constructed ENIIRA strains are evaluated for the correctness of the introduced mutations and the phenotypes expected are confirmed using all the methods described in Example 1. In this regard, Family A strains such as χ12517 commence to lyse as they attempt to grow in any medium lacking DAP and D-alanine such that cultures completely lyse within several hours after transfer from a permissive growth medium to a non-permissive growth condition in the absence of DAP and D-alanine (FIG. 3). Family B strains, however, will undergo several cell divisions after transfer to non-permissive media since the enzymes for synthesis of DAP and D-alanine that cease to be synthesized are diluted by half at each cell division until their concentration is insufficient to enable synthesis of sufficient DAP and D-alanine to enable peptidoglycan synthesis. This behavior is observed for χ12518 (FIG. 3) during the first several hours of growth under non-permissive growth conditions. However, for cultures tested under high cell density lysis leads to destruction of the peptidoglycan cell wall layer to release its constituents DAP and D-alanine that are now free to be incorporated into peptidoglycan in surviving cells to cause an increase in viable cell titer. If, however, cultures of χ12518 are inoculated into non-permissive growth conditions at densities of 1×10⁵ CFU or lower, death by lysis continues until the culture is devoid of viable cells.

h. Discussion

As each of the Sal-Adj/ENIIRA strains described above is constructed its properties are being fully validated. The abilities of these strains in comparison to parent and control strains to stimulate NF-kB production in HEK cells having different TLR and Nod factors were evaluated (Example 3). These results also led to synthesis of strains with combinations of mutations and the conduct of animal studies to determine which combination of mutations yields a Sal-Adj/ENIIRA strain(s) with greatest ability to enhance immune responses to Ova (Example 4) and protection against Mtb. 3 (Example 5).

Example 3. Evaluation of Sal-Adj/ENIIRA Strains to Activate Synthesis of NF-kB Production in HEK293 Cells

Strains constructed in the Example 2 research were initially tested and compared for their ability to interact with and stimulate HEK293 cells with TLR1, TLR2, TLR4 (two types), TLR5 (two types), TLR6, TLR9, Nod1 and Nod2 using methods described in Example 1 M&M. Most evaluations have been with Family A strains since they commence to lyse immediately upon placing in non-permissive conditions in the absence of arabinose (see FIG. 3 above) such that lysis would occur during and after invasion of cells. In this regard, Family B strains are able to undergo several cell divisions after placing in non-permissive conditions in the absence of arabinose such that they will not be lysing after adding to HEK cells. This is because Salmonella cell division in infected cells ranges from 8 to more than 20 hours depending on cell type compared to less than an hour for most in vitro growth conditions. These inferences are validated by the results presented in FIG. 2 above in which the Family A strain χ9052 is more active in recruiting a response in HEK cells with the internal Nod1 receptor than is the Family B strain χ12499 (See FIG. 2B).

Results observed in evaluation of several Family A and Family B strains in assays of NFκB recruitment in HEK cells displaying TLR4 are presented in FIG. 5. In these studies, we varied the multiplicity of infection (MOI) of ENIIRA strains to HEK cells from 0.001:1 to 10:1. Based on these and other evaluations, Family B strains such as χ12518 were better recruiters than Family A strains such as χ12517. In addition, removal of the O-antigen or parts of the LPS core further enhanced recruitment of NFκB (compare χ12518 versus χ12542 and χ12543. All ENIIRA strains were more active in TLR4 activation than the wild-type parent χ3761.

Results observed in evaluation of several Family A and Family B strains in assays of NFκB recruitment in HEK cells displaying TLR5 are presented in FIG. 6. We also include data for control strains in FIG. 6 that evaluate mutant strains derived from the wild-type parent strain χ3761. The only strain unable to activate TLR5 is χ9028 that is unable to synthesize flagellin or flagella due to the ΔfliC2426 and ΔfljB217 mutations. Thus, all the strains with the ΔfliC180 mutation activate TLR5 even though many are non-motile due to fljB deletion mutation.

As noted above in reference to the FIG. 2 data, Family A strains are better at activating Nod1 than are Family B strains due to their more rapid lysis after entry into cells. Nevertheless, Family B strains with either ΔwaaC41 or ΔwaaG42 with or without the ΔpagP81::P_(lpp) lpxE deletion-insertion mutation are proficient in activating Nod1 and Nod2 (FIG. 7).

Example 4. Evaluation of Sal-Adj/ENIIRA Strains by s.c., i.n. And Oral Routes with s.c. Administration of Ova Suspended in Buffered Saline or Alum and Monitoring Antibody Responses to Ova

Because of total avirulence, we concentrated on testing the adjuvant activity of the Family A parental strain χ9052. The results presented in FIG. 8 reveal very clearly that co-administration of the ENIIRA strain with Ova was far superior to administering Ova with the standard Alum adjuvant in eliciting serum IgG antibodies against Ova. Studies with Family B strains such as with the avirulent derivative χ12544 (Table 5) have just commenced with the expectation that they will likely be better than Family A strains in these assays measuring enhanced antibody production to subunit vaccines. In this regard, because of the ability of Family B strains to undergo several rounds of cell division in vivo prior to lysis, we expect them to be effective at lower doses that needed for Family A strains

Example 5. Evaluation of the Ability of ENIIRA Strains to Augment Ability of BCG to Diminish Infection of Mice with M. tuberculosis H37RV (Mtb) and Whether Also Enhances the Ability of a PIESV Construct Delivering Mtb Protective Antigens to Further Protect Against Mtb Infection and Proliferation

Three experiments have been conducted in which ENIIRA strains have been administered in combination with M. bovis BCG, which is currently the only vaccine approved for human use to prevent infections by M. tuberculosis and development of TB. In all of these experiments, BCG was administered s.c. with 5×10⁴ CFU of BCG, either alone or in combination with ENIIRA strains and/or the PIESV χ12068(pYA4891). All immunizations/inoculations were given once. All experiments included a group of mice that were administered 100 μl of phosphate-buffered saline PBS on Day 0; these were unimmunized control mice. Thirty-five days after immunization, all mice were challenged with a low-dose aerosol of virulent M. tuberculosis H37Rv, such that each mouse received 50-100 bacteria per lung. On day 28 in Experiments 15 and 20 (FIGS. 10 and 11), blood was collected from all mice to determine antibody titers to the mycobacterial Antigen 85A, which is produced by both BCG and M. tuberculosis. In Experiments 15 and 20, two mice from each group were euthanized on day 28, their lungs and spleens homogenized and processed for flow cytometry, to evaluate T-cell responses to Ag85A and, in Experiment 20, to two M. tuberculosis antigens (ESAT-6 and CFP-10) produced by the PIESV χ12068(pYA4891) construct. Approximately 6 weeks after challenge, mice were euthanized, lungs and spleens were removed, homogenized and plated for CFU determinations.

In Experiment 14 (FIG. 9), the ENIIRA strain was χ12499 (Family B) (ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB), which was administered by itself or in combination with BCG by either s.c. administration of 1×10⁵ CFU of χ12499 or by i.v. administration of 1×10⁴CFU of χ12499. Only M. tuberculosis CFU determinations were done in this experiment and we found that mice that had been immunized with the combination of BCG+χ12499 administered i.v. had CFUs of approximately 2×10⁴ in their spleens, compared to titers of 1×10⁵ CFU in the spleens of mice immunized with BCG alone and titers of 3×10⁵ CFU in the spleens of the unimmunized control mice, suggesting that administration of χ12499 by the i.v. route enhanced the protection afforded by BCG (FIG. 9). In the lungs of these mice, co-administration of χ12499 (i.v.) with BCG yielded CFU titers that were lower than the unimmunized control mice (5×10⁵ for immunized mice compared to 2×10⁶ CFU for unimmunized mice), but were not as low as mice immunized with BCG alone (4×10⁵ CFU).

In Experiment 15 (FIG. 10), co-administration of BCG with two ENIIRA strains (χ12517 (Family A) (Δalr-3 ΔdadB4 ΔasdA33 ΔfliC180 ΔpagP81::P_(lpp) lpxE ΔpagL7 ΔlpxR9) and χ12518 (Family B) (ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB ΔfliC180 ΔpagP81::P_(lpp) lpxE ΔpagL7 ΔlpxR9)), both delivered by i.v. (5×10⁴ CFU) or the PIESV χ12068(pYA4891) (ΔP_(murA25)::TT araC P_(araBAD) murA ΔasdA27::TT araC P_(araBAD) c2 Δ(wza-wcaM)-8 Δpmi-2426 ΔrelA197::araC P_(araBAD) lacI TT ΔrecF126 ΔsifA26 ΔwaaL46 ΔpagL12:TT araC P_(araBAD) waaL+M. tuberculosis antigens Ag85A, ESAT-6 and CFP-10)), delivered by i.v. (5×10⁴ CFU) was evaluated. In this experiment, the CFU titers of M. tuberculosis in the lungs of mice immunized with the combination of BCG+χ12068(pYA4891) were 5.5×10⁴ CFU, compared to titers of 2×10⁵ CFU in BCG-immunized mice and 2×10⁶ CFU in unimmunized control mice. Mice immunized with BCG+χ12517 or BCG+x12518 had titers that were the same as mice immunized with BCG alone (2×10⁵ CFU). In the spleens of the mice in this experiment (FIG. 10), the titers of M. tuberculosis in mice immunized with the combination of BCG+χ12518 were 1.5×10³ CFU, compared to titers of 4×10⁴ CFU in mice immunized with BCG+χ12068(pYA4891) and approximately 6×10⁴CFU in mice immunized with BCG alone or BCG+χ12517. The titers in the unimmunized control mice were 2.5×10⁵ CFU. Flow cytometry analyses of the percentage of total T cells and dendritic cells (DCs) secreting Interferon-γ (IFN-γ) in response to Ag85A indicated that the highest percentage of IFN-γ-secreting T cells and DCs were from the spleens of mice immunized with BCG+χ12518, although all immunized mice had higher percentages of IFN-γ-secreting T cells and DCs in their spleens than the unimmunized control mice.

In Experiment 20 (FIG. 11), we repeated the comparisons of mice immunized with BCG+χ12068(pYA4891), delivered i.v., and BCG+χ12518, delivered i.v., compared to mice immunized with BCG alone and to unimmunized mice. We also compared mice immunized with BCG+χ12068(pYA4891)+χ12518, both delivered by i.v. injection of a total of 5×10⁴CFU and mice immunized with BCG+χ12068(pYA4891), delivered by intranasal (i.n.) administration of 1×10⁵ CFU, plus χ12518 delivered by i.v. injection (5×10⁴CFU). We do not yet have the titers of M. tuberculosis in the spleens of the mice form this experiment. FIG. 11 shows the CFU titers of M. tuberculosis H37Rv in the lungs. Mice vaccinated with BCG alone or BCG+χ12068(pYA4891) had titers that were more than one log lower than the CFU titers in the unimmunized mice. Mice immunized with the combination of BCG+χ12068(pYA4891)+χ12518 (both delivered i.v.) had titers that were two logs lower than the titers in the unimmunized mice. Mice that were immunized with the combination of BCG+χ12518 or BCG+χ12068(pYA4891) (delivered i.n.)+χ12518 (delivered i.v) also had significantly lower CFU titers in their lungs, compared to unimmunized mice. These collective results were most surprising since the highly significant enhancement of the efficacy of BCG in reducing Mtb multiplication has never been previously observed. Thus, the ability of ENIIRA strains to enhance the effectiveness of BCG vaccination could have a profound global benefit in prevention of M. tuberculosis infections. This is highly significant since tuberculosis is now the number one cause of global deaths from any infectious disease. We have determined total IgG antibody responses in the sera of these mice to Ag85A, ESAT-6 and CFP-10. We found that IgG levels to Ag85A were higher in mice immunized with combinations of BCG and ENIIRA or PIESV χ12068(pYA4891), compared to mice immunized with BCG alone, with the highest levels in mice immunized with BCG+χ12068(pYA4891)+χ12518, regardless of whether χ12068(pYA4891) was delivered by i.v. or by i.n. inoculation. We also found that mice immunized with BCG+χ12068(pYA4891), delivered i.n., +χ12518 had the highest levels of total IgG against ESAT-6 and CFP-10. We also conducted flow cytometric analyses on the spleens and lungs of mice in this experiment. In the spleens, mice immunized with the combination of BCG+χ12068(pYA4891) delivered i.n.+χ12518 had the highest percentages of IFN-γ-secreting CD4⁺ T cells responding to ESAT-6, compared to mice in each of the other groups. Mice immunized with that combination also had the highest percentages of IFN-γ-secreting CD8⁺ T cells responding to Ag85A, ESAT-6 and CFP-10. In the lungs of these mice, immunization with the combination of BCG+χ12068(pYA4891) delivered i.n.+χ12518 induced the highest or close to highest levels of IFN-γ-secreting CD8⁺ T cells in response to Ag85A, ESAT-6 or CFP-10 among all of the groups of immunized mice. In both lungs and spleens, mice immunized with the combinations of BCG+χ12518 strains or BCG+χ12068(pYA4891) or BCG+χ12518+χ12068(pYA4891) all induced higher percentages of IFN-γ-secreting CD4⁺ and CD8⁺ T cells than mice immunized with BCG alone.

The results from the three experiments described above demonstrate that co-administration of BCG with ENIIRA strains plus χ12068(pYA4891) enhances the ability of BCG to protect mice against aerosol challenge with M. tuberculosis and that co-administration enhances both antibody and T-cell responses that is likely to contribute to protection against challenge. If found to be true in humans, the impact will be highly significant.

Splenomegaly: In experiments in which mice were immunized subcutaneously with M. bovis BCG alone or in combination with ENIIRA strains or the PIESV χ12068(pYA4891) or both an ENIIRA strain and χ12068(pYA4891), splenomegaly was observed in mice immunized with BCG and an ENIIRA strain. In Experiment 14, where the ENIIRA strain was χ12499 (Family B), 2 out of 9 mice immunized with χ12499 (delivered s.c.) alone, 2 out of 10 mice immunized with the combination of BCG+χ12499 (both delivered s.c) and 7 out of 8 mice immunized with the combination of BCG (delivered s.c.)+χ12499 (delivered i.v.) had significantly enlarged spleens, compared to the spleens of control mice (PBS administered s.c) and mice immunized with BCG alone, in which none of the mice had enlarged spleens. In Experiment 15, in which mice were immunized with BCG alone (delivered s.c.) or BCG (delivered s.c.) in combination with the PIESV χ12068(pYA4891) or Family A ENIIRA strain χ12517 or Family B ENIIRA strain χ12518 (each administered i.v), only mice immunized with the combination of BCG+χ12518 had enlarged spleens. In Experiment 20, mice were immunized with BCG alone (delivered s.c.) and in combination with the PIESV χ12068(pYA4891), delivered i.v., the Family B ENIIRA strain χ12518 (delivered i.v.), χ12068(pYA4891) and χ12518 (both delivered i.v.) or χ12068(pYA4891), delivered intranasally, and χ12518, delivered i.v. All groups of mice immunized with χ12518 (which was always administered i.v.) had mice with enlarged spleens. At present, we do not know why some mice in these three experiments developed enlarged spleens.

However, these results are in accord with other observations that indicate that the Family B strain χ12518 (ΔP_(asdA55)::TT araC P_(BAD) asd Δalr-3 ΔP_(dadB66)::TT araC P_(BAD) dadB ΔfliC180 ΔpagP81::P_(lpp) lpxE ΔpagL7 ΔlpxR9) is possibly too inflammatory because it multiplies too many cell divisions prior to lysis. Although this can be addressed by using lower doses, we are now testing the benefits of including the ΔwaaL46 (χ12544) and ΔrecA62 mutations or both on ensuring complete attenuation while retaining the beneficial adjuvant activities.

Example 6. Modification of ENIIRA Strains to Display PAMPS that Activate Innate Immune Receptors Displayed by Diverse Bacterial, Viral, Fungal and Parasite Pathogens

Our group has displayed capsular polysaccharides specified by genes from gram-negative and gram-positive bacterial species on the surface of Salmonella strains. We have also expressed lipo-proteins and protein appendages encoded by genes from diverse pathogens. In addition, since the ENIIRA strains are designed to lyse, they can liberate plasmids engineered to display single-stranded and double-stranded RNAs as displayed by and serving as PAMPS for RNA viruses. It is thus possible to modify ENIIRA strains to induce innate immune responses that could differ and be more appropriate and efficacious in enhancing immunity to be induced by a diversity of vaccines targeting prevention of infection by diverse bacterial, viral, fungal and parasite pathogens.

Example 1. Non-Specific Protection Against Infection by Other Pathogens

It has been found that administration of the Sal-Adj/ENIIRA constructs induces low-level protective immunity to challenge of unvaccinated animals to various bacterial, viral and parasite pathogens as revealed by the data in Table 3 in which the empty vector PIESV strains are representative of Family C ENIIRA strains. These results are impactful in the protection, in some examples, of military personnel and civilians against a biothreat as well as to contend with epidemics. These ENIIRA strains will also have utility in augmenting levels of protective immunity of subunit and killed vaccines and even of attenuated vaccines that do not induce robust protective immunity of long duration, such as BCG. It can also be expected that the level of innate immunity induced by administering ENIIRA strains will be of reasonably long duration. In fact, these strains will probably be effective in inducing memory innate immune responses.

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What is claimed is:
 1. An adjuvant for the enhancement of vaccine efficacy, the adjuvant comprising an.
 2. The adjuvant of claim 1, wherein the bacterial pathogen is a Salmonella spp.
 3. The adjuvant of claim 1 or 2, wherein the adjuvant comprises an attenuated Salmonella typhimurium (S. typhimurium) bacterium, comprising (a) one or more mutations facilitating lysis in vivo, comprising (i) ΔP_(murA)::TT araC P_(araBAD) murA, (ii) ΔasdA, (in) ΔP_(asdA)::TT araC P_(araBAD) asdA, (iv) Δalr, (v) ΔdadB, or (vi) ΔP_(dadB66)::TT araC P_(araBAD) dadB, or a combination thereof; (b) one or more mutations to enhance recruitment of innate immunity comprising (i) ΔpagP::P_(lpp) lpxE, (ii) ΔpagL, or (iii) ΔlpxR, ΔarnT, ΔeptA, ΔwaaC. ΔwaaG, ΔwaaL or a combination thereof; (ii) ΔP_(stc)::P_(murA) stc, (ii) ΔstcABCD, (iii) ΔP_(saf)::P_(murA) saf, or (iv) ΔsafABCD, or a combination thereof, or (c) a mutation eliminating or diminishing effective immunogenicity, comprising ΔsifA; or a combination of at least two of (a), (b), or (c).
 4. A composition comprising the adjuvant of any of claims 1-3 and a pharmaceutically acceptable carrier.
 5. A method of augmenting induction of protective immunity by a vaccine, the method comprising administering an immune response enhancing amount of the adjuvant of any of claims 1-3.
 6. The method of claim 5, wherein the vaccine comprises a subunit, killed, live attenuated, or vectored vaccine.
 7. An adjuvant composition, the composition comprising the bacterium of claim
 3. 8. A method of providing an induced protective immunity to a pathogen, comprising co-administering an adjuvant composition, comprising the adjuvant of any of claims 1-3, and co-administering a vaccine composition comprising an antigen to the pathogen.
 9. A method of providing an induced protective immunity to a pathogen, comprising administering M. bovis BCG and co-administering an adjuvant composition comprising the adjuvant of any of claims 1-3.
 10. The method of claim 9, further comprising co-administering a PIESV strain engineered to deliver one or more Mtb protective antigens.
 11. The method of claim 10, wherein the PIESV strain is PIESV χ12068(pYA4891).
 12. A method of providing an induced protective immunity to a pathogen, comprising administering M. bovis BCG and co-administering a PIESV strain engineered to deliver one or more Mtb protective antigens.
 13. The method of claim 12, wherein the PIESV strain engineered is PIESV χ12068(pYA4891).
 14. The method of either of claim 12 or 13, further comprising co-administering an adjuvant composition comprising the adjuvant of any of claims 1-3.
 15. The method of claim 13, wherein the adjuvant comprises an attenuated Salmonella typhimurium (S. typhimurium) bacterium, comprising (a) one or more mutations facilitating lysis in vivo, comprising (i) ΔP_(murA)::TT araC P_(araBAD) murA, (ii) ΔasdA, (iii) ΔP_(asdA)::TT araC P_(araBAD) asdA, (iv) Δalr, (v) ΔdadB, or (vi) ΔP_(dadB66)::TT araC P_(araBAD) dadB, or a combination thereof; (b) one or more mutations to enhance recruitment of innate immunity comprising (i) ΔpagP::P_(lpp) lpxE, (ii) ΔpagL, or (iii) ΔlpxR, ΔarnT, ΔeptA, ΔwaaC. ΔwaaG, ΔwaaL or a combination thereof; (ii) ΔP_(stc)::P_(murA) stc, (ii) ΔstcABCD, (in) ΔP_(saf)::P_(murA) saf, or (iv) ΔsafABCD, or a combination thereof, or (c) a mutation eliminating or diminishing effective immunogenicity, comprising ΔsifA; or a combination of at least two of (a), (b), or (c). 